Differentiation of monocytic U937 cells under static magnetic field exposure

We present here a morphological, cytochemical and biochemical study of the macrophagic differentiation of human pro-monocytic U937 cells exposed to moderate intensity (6 mT) static magnetic fields (MF). It was found that the following substances induced differentiation in U937 cells to a progressively lower degree: 50 ng/mL 12-0-tetradecanoyl-13-phorbol acetate (TPA), low concentration of glutamine (0,05 mM/L), 10% dimethyl sulfoxide (DMSO) and 100 mM/L Zn++. Differentiated U937 cells shift from a round shape to a macrophage-like morphology, from suspension to adhesion growth and acquire phagocytotic activity, the cytoskeleton adapting accordingly. Exposure to static MF at 6 mT of intensity decreases the degree of differentiation for all differentiating molecules with a consequent fall in cell adhesion and increased polarization of pseudopodia and cytoplasmic protrusions. Differentiation alone, or in combination with exposure to static MFs, affects the distribution and quantity of cell surface sugar residues, the surface expression of markers of macrophage differentiation, and phagocytotic capability. Our results indicate that moderate-intensity static MFs exert a considerable effect on the process of macrophage differentiation of pro-monocytic U937 cells and suggest the need for further studies to investigate the in vivo possible harmful consequences of this.     

Biological effects of 6 mT static magnetic fields: a comparative study in different cell types

The present work was a comparative study of the bio-effects induced by exposure to 6 mT static magnetic field (MF) on several primary cultures and cell lines. Particular attention was dedicated to apoptosis. Cell viability, proliferation, intracellular Ca(2+) concentration and morphology were also examined. Primary cultures of human lymphocytes, mice thymocytes and cultures of 3DO, U937, HeLa, HepG2 and FRTL-5 cells were grown in the presence of 6 mT static MF and different apoptosis-inducing agents (cycloheximide, H(2)O(2), puromycin, heat shock, etoposide). Biological effects of static MF exposure were found in all the different cells examined. They were cell type-dependent but apoptotic inducer-independent. A common effect of the exposure to static MF was the promotion of apoptosis and mitosis, but not of necrosis or modifications of the cell shape. Increase of the intracellular levels of Ca(2+) ions were also observed. When pro-apoptotic drugs were combined with static MF, the majority of cell types rescued from apoptosis. To the contrary, apoptosis of 3DO cells was significantly increased under simultaneous exposure to static MF and incubation with pro-apoptotic drugs. From these data we conclude that 6 mT static MF exposure interfered with apoptosis in a cell type- and exposure time-dependent manner, while the effects of static MF exposure on the apoptotic program were independent of the drugs used.     

Regulation of the growth and differentiation of a human monocytic cell line by lymphokines. I. Induction of superoxide anion production and chemiluminescence

The U937 human monocytic cell line was studied to determine its ability to generate a respiratory burst after stimulation with phorbol myristate acetate (PMA) or opsonized zymosan. U937 cells cultured in normal medium produced virtually no superoxide anion or chemiluminescence in response to either stimulus. In contrast, U937 cells cultured in medium containing soluble factors from activated lymphocytes produced significant O2- and chemiluminescence when stimulated with PMA or opsonized zymosan. The chemiluminescence in response to PMA was maximal in U937 cells precultured with these soluble factors for 3 days, whereas maximal responsiveness to opsonized zymosan was not observed until 5 to 6 days of lymphokine exposure. Although this ability to generate a respiratory burst persisted for a number of days in U937 cells that were subsequently recultured in normal medium, this responsiveness was gradually lost in the continued absence of these factors. The data indicate that the U937 monocytic cell line can be activated or induced to differentiate by soluble factors released by activated lymphocytes. In the process, these cells acquire the ability to generate a respiratory burst. The U937 cell line may serve as a useful model for the study of the ontogeny and regulation of the respiratory burst during human monocytic differentiation.     

Differentiation and dedifferentiation of the human monocytic leukemia cell line, U937.

U937 cells were differentiated into macrophages after being treated with 12-o-tetradecanoyl-phorbol-13-acetate (TPA) for the first two days and dedifferentiated with daily medium renewal for 10 days. Cell proliferation slowed down and the number of cells reached the maximum level on day 2. By day 4, all of the cells had spread and attached firmly to the culture dish, and more than 90% of the cells expressed the Fc-receptor and produced superoxide anion. From there on, the number of adherent, living cells decreased gradually to about half the initial count. Most of the cells eliminated from the culture by cell death were in the S phase at the time of TPA treatment. After day 8, the number of cells expressing macrophage-specific phenotypes gradually decreased, cell adhesion was weakened, and at the same time, DNA synthesis was initiated anew. The cells became round and began to proliferate as floating cells on days 9 to 10, and thereafter they became sensitive to the second round of TPA treatment. On the basis of all the results taken together, it is suggested that fully differentiated U937 cells were dedifferentiated after being cultured with frequent medium renewal.     

Autogenous production of interferon-beta switches on HLA genes during differentiation of histiocytic lymphoma U937 cells

The expression of class I HLA genes was measured during the in vitro differentiation of human U937 lymphoma cells towards macrophages. Following the onset of differentiation by phorbol myristate acetate the levels of cytoplasmic mRNA that hybridized with a [32P]HLA-B cDNA probe increased by a factor of nine. Elevation in HLA mRNA accumulation was followed by an increase in the rate of synthesis of HLA proteins and also by a dramatic increase in class I HLA cell surface antigen expression, as shown by cytofluorimetric analysis. The elevation in HLA mRNA and surface antigens could be prevented by adding antibodies against human interferon-beta (IFN-beta) to the culture medium at the onset of differentiation. Interferon antiviral activity was detected in the medium of differentiated U937 cells. The same anti-IFN-beta antibodies prevented the increase in (2'-5')oligo(A) synthetase activity which also takes place in differentiating U937 cells. Accumulation of the IFN-induced (2'-5')oligo(A) synthetase in U937 cells is preceded by an increase in its specific 1.6-kb mRNA as shown by hybridization to cloned (2'-5')-oligo(A) synthetase cDNA. The enzyme was preferentially found in the nuclear fraction of differentiating U937 cells. We suggest that an autogenous production of interferon-beta by the differentiating cells, switches on expression of the class I HLA genes as well as that of the (2'-5')oligo(A) synthetase.     

Ectopic expression of CXCR5/BLR1 accelerates retinoic acid- and vitamin D(3)-induced monocytic differentiation of U937 cells

The product of the blr1 gene is a CXC chemokine receptor (CXCR5) that regulates B lymphocyte migration and has been implicated in myelomonocytic differentiation. The U937 human leukemia cell line was used to study the role of blr1 in retinoic acid-regulated monocytic leukemia cell growth and differentiation. blr1 mRNA expression was induced within 12 hr by retinoic acid in U937 cells. To determine whether the early induction of blr1 might regulate inducible monocytic cell differentiation, U937 cells were stably transfected with blr1 (U937/blr1 cells). Ectopic expression of blr1 caused no significant cell cycle or differentiation changes, but caused the U937/blr1 cells to differentiate faster when treated with either retinoic acid or 1alpha,25-dihydroxyvitamin D(3). Treated with retinoic acid, U937/blr1 cells showed a greater increase in the percentage of CD11b expressing cells than vector control cells. Retinoic acid also induced a higher percentage of functionally differentiated blr1 transfectants as assessed by nitroblue tetrazolium reduction. U937/blr1 cells underwent moderate growth inhibition on treatment with retinoic acid. Similar results occurred with 1alpha,25-dihydroxyvitamin D(3). Because blr1 was induced early during cell differentiation and because its overexpression accelerated monocytic differentiation, it may be important for signals controlling cell differentiation.     

The influence of cell shape on the induction of functional differentiation in mouse mammary cells in vitro

Summary To define more clearly the in vitro conditions permissive for hormonal induction of functional differentiation, we cultured dissociated normal mammary cells from prelactating mice in or on a variety of substrates. Cultivation of an enriched epithelial cell population in association with living adult mammary stroma in the presence of lactogenic hormones resulted in both morphological and biochemical differentiation. This differentiation, however, was not enhanced over that seen when the cells were associated with killed stroma, provided that the killed stroma had a flexibility similar to that of the living stroma. Cells cultured in inflexible killed stroma usually did not differentiate. Cells cultured within the flexible environment of a collagen gel, but removed from the gas-medium interface, differentiated in a manner similar to those cultured in flexible stroma. Cells cultured on the surface of an attached collagen gel were squamous, and their basolateral surfaces were sequestered from the medium; they did not differentiate. Cells cultured on floating collagen gels were cuboidal-columnar, with basolateral surfaces exposed to the medium, and showed good functional differentiation. Cells cultured on inflexible floating collagen gels were extremely flattened and had exposed basolateral surfaces, and showed no evidence of functional differentiation. We infer that assumption of cuboidal to columnar shapes similar to those of mammary cells in vivo may be important to the induction of functional differentiation in vitro. The additional requirement of basolateral cell surface exposure also is important. This work was supported by U.S. Public Health Service Grants CA-05045 and CA-09041 from the National Cancer Institute, Bethesda, MD.     

Augmentation of Interferon Production after Cell-Differentiation of U937 Cells by TPA

Persistent infections with mumps virus were established in several human lymphoid cells of T-cell origin (Molt-4, TALL-1, and CCRF-CEM) and human monocyte cells (U937 and THP-1). 2′,5′-Oligoadenylate synthetase (2–5AS) activity was demonstrated to be only slightly induced by interferon (IFN) or TPA (12-O-tetradecanoyl-phorbol-13-acetate) treatment in these cells. Treatment of the persistently infected cells with IFN or TPA did not stimulate an increase in the amount of synthetase mRNA. Induction of cell differentiation and augmentation of IFN production by TPA were demonstrated in U937 cells persistently infected with mumps virus (U937-MP). Similar results for IFN production were obtained from differentiated U937 cells. It is suggested that cell differentiation of U937 cells might be associated with the development of IFN inducibility.     

Cell shape and plasma membrane alterations after static magnetic fields exposure

The biological effects of static magnetic fields (MFs) with intensity of 6 mT were investigated in lymphocytes and U937 cells in the presence or absence of apoptosis-inducing drugs by transmission (TEM) and scanning (SEM) electron microscopy. Lectin cytochemistry of ConA-FITC conjugates was used to analyze plasma membrane structural modifications. Static MFs modified cell shape, plasma membrane and increased the level of intracellular [Ca++] which plays an antiapoptotic role in both cell types. Modifications induced by the exposure to static MFs were irrespective of the presence or absence of apoptotic drugs or the cell type. Abundant lamellar-shaped microvilli were observed upon 24 hrs of continuous exposure to static MFs in contrast to the normally rough surface of U937 cells having numerous short microvilli. Conversely, lymphocytes lost their round shape and became irregularly elongated; lamellar shaped microvilli were found when cells were simultaneously exposed to static MFs and apoptosis-inducing drugs. In our experiments, static MFs reduced the smoothness of the cell surface and partially impeded changes in distribution of cell surface glycans, both features being typical of apoptotic cells. Cell shape and plasma membrane structure modifications upon static MFs exposure were time-dependent. Lamellar microvilli were clearly observed before the distortion of cell shape, which was found at long times of exposure. MFs exposure promoted the rearrangement of F-actin filaments which, in turn, could be responsible for the cell surface modifications. Here we report data that support biological effects of static MFs on U937 cells and human lymphocytes. However, the involvement of these modifications in the onset of diseases needs to be further elucidated.     

Chromium chloride inhibits oxidative stress and TNF-alpha secretion caused by exposure to high glucose in cultured U937 monocytes.

Chromium supplementation has been proposed to promote the action of insulin and the lowering of blood glucose levels in diabetic patients. However, the molecular mechanism by which chromium increases insulin sensitivity is not known. Using U937 monocytes as a cell culture model, this study demonstrates that chromium inhibits the secretion of TNF-alpha, a cytokine known to inhibit the sensitivity and action of insulin. U937 cells were cultured with high levels of glucose (mimicking diabetes) in the presence or absence of chromium chloride in the medium at 37 degrees C for 24 h. This study demonstrates that chromium supplementation prevents the increase in TNF-alpha levels and oxidative stress caused by the high levels of glucose in cultured U937 monocytic cells. Similarly, chromium supplementation prevented elevated TNF-alpha secretion and lipid peroxidation levels in H(2)O(2)-treated U937 cells. This study demonstrates for the first time that chromium supplementation inhibits TNF-alpha secretion in U937 monocytes cultured in high-glucose medium, which appears to be mediated by its antioxidative effect. This provides evidence for a novel molecular mechanism by which chromium supplementation may increase insulin sensitivity and glycemic control in diabetic patients.     

Human mononuclear phagocyte-associated antigens: II. Lymphokine-inducible antigens on the macrophage cell line,U937

We have utilized the U937 macrophage cell line as a model system for analysis of human mononuclear phagocyte (MNP) differentiation. In addition to expressing membrane antigens shared with other MNP, U937 possesses an intrinsic ability to become “activated” upon exposure to lymphokines. A heteroantiserum produced against lymphokine-stimulated U937 (anti-U937L) was utilized to detect acquired or inducible membrane antigens expressed on “activated” U937. Absorption of this antiserum to remove antibodies to nonstimulated U937 (U937N) did not remove the reactivity of anti-U937L/U937N to lymphokine-stimulated U937 as determined by an ¹²⁵I-protein A radioimmunoassay. The lymphokine-inducible antigens were not detectable on resident, human peritoneal macrophages. In addition to expression of lymphokine-inducible antigens, treated U937 cells displayed alterations in both morphology and functional activity (antibody-dependent cellular cytotoxicity). Kinetic analysis of lymphokine-stimulated U937 indicated that antigen expression occurred as early as 1–2 hr after lymphokine exposure, plateauing at 16–18 hr of stimulation. The inducible antigens were susceptible to proteolytic degradation and expression was blocked by inhibitors of protein synthesis. Inducible antigens detectable by anti-U937L/U937N did not result from the expression of cryptic or buried membrane antigens. Thus, the U937 cell line can be utilized for production of antibodies useful in analysis of membrane antigen expression during differentiation within the MNP system.     

Respiratory syncytial virus infection of human mononuclear phagocytes stimulates synthesis of platelet-activating factor

Production of platelet-activating factor 1-O-alkyl-2-acetyl-sn-glycero-3- phosphocholine (PAF), a potent mediator of inflammation, by mononuclear phagocytes varies with their stage of cellular differentiation and the nature of the eliciting stimulus. The human monocytic cell line U937 can be induced to differentiate to a macrophage-like cell following phorbol myristate acetate exposure, and after differentiation, these cells efficiently support replication of respiratory syncytial virus (RSV). U937 cells induced to differentiate with phorbol myristate acetate demonstrated a time-dependent decrease in PAF synthesis. RSV infection of these differentiated U937 cells caused a sustained stimulation of PAF synthesis that paralleled viral replication and was dependent on infectious virus. Virus increased the activity of lyso-PAF:acetyl-CoA acetyl-transferase (PAF acetyltransferase) in cell lysates, thus enhancing the anabolic pathway of PAF synthesis without altering the activity of PAF acetylhydrolase, which regulates PAF catabolism. RSV infection of human monocytes also caused a marked increase in [3H] monocytes also caused to uninfected monocytes. Thus, virus infection serves as a novel stimulus to induce PAF synthesis in human mononuclear phagocytes and suggests that increased PAF production may have a critical role in the inflammatory response to RSV.     

Differential effects of ETYA, a PUFA-analogue, on prostate (PC3) and monoblastoid U937 ultrastructure; lack of correlation with reduced proliferation.

ETYA (5,8,11,14-eicosatetraynoic acid), a polyunsaturated fatty acid analogue, inhibits proliferation of PC3 and U937 cells and induces a limited differentiation in U937 cells. Human prostate PC3 cells cultured for 72 h with 40 microM ETYA in fetal calf serum contained putative lipofuscin bodies, myelin figures and mitochondria with damaged cristae and matrices. These changes were absent from human U937 monoblastoid cells incubated with ETYA in CPSR3, a semipurified serum replacement. U937 cells cultured with ETYA in fetal calf serum contained occasional lipofuscin bodies, while PC3 cells cultured in CPSR3 exhibited all of the changes described. ETYA reduced the oxygen consumption of both cell lines. Therefore we conclude: (a) The response to ETYA by cells of dissimilar developmental origin is not identical; (b) unidentified serum components can augment potential ETYA-induced oxidative stress-responses of cells; (c) inhibition of U937 proliferation by ETYA does not depend upon the morphologic changes seen in PC3 cells, which resemble sequelae of oxidative stress with excess free radicals; and (d) rapid ETYA-induced inhibition of oxygen consumption in both cell lines implies a reduced synthesis of ATP that could contribute to the reversible impairment of cellular proliferation.     

Shigella flexneri YSH6000 induces two types of cell death, apoptosis and oncosis, in the differentiated human monoblastic cell line U937

Shigella flexneri, but not a non-invasive mutant derivative rapidly induced cell death in human monoblastic U937 cells as well as in differentiated cells pretreated with interferon-gamma (IFN gamma) or retinoic acid (RA). We investigated the morphological and biochemical characteristics of bacterial invasion-induced cell death in these differentiated U937 cells. IFN gamma-differentiated cells showed morphological changes typical of apoptosis and their DNA was cleaved giving a ladder-like electrophoretic pattern after infection by Shigellae. In contrast, swelling of the cytoplasm and blebbing of the plasma membrane were observed in RA-differentiated and undifferentiated cells invaded by the bacteria. No condensation of nuclei was observed in these cells by light microscopy, and no internucleosomal fragmentation of DNA was detected on agarose gels, which resembled the features of oncosis. Furthermore, cleavage of poly(ADP-ribose) polymerase, a substrate for apoptotic caspases, was seen only in IFN gamma-pretreated cells but not in RA-pretreated or undifferentiated cells. These findings suggested that virulent Shigella flexneri induces distinct types of cell death in U937 cells depending on their differentiation state.     

Investigation on the effect of static magnetic field up to 30 mT on viability percent,proliferation rate and IC50 of HeLa and fibroblast cells

Abstract

We have investigated the effects of static magnetic field (SMF) on the viability of the human cervical cancer (HeLa) cell line and fibroblast cells. The cells were cultured in DMEM medium and treated several times (24, 48,72 and 96?h) and at several intensities (5, 10, 20 and 30?mT) of magnetic field (MF). The cytotoxicity and cell viability percent in treated cells were performed using MTT assay by evaluating mitochondrial dehydrogenase activity. The MF ability on inducing cell death or inhibiting biochemical function was reported as cell death percent. The results showed that the increase of MF intensity and the time that cells were exposed to this treatment increased sharply cell death percent and proliferation rate in HeLa cell compare to fibroblast cells. Our data suggest that SMF biological effects on cell death were different in our selected targets. Cell type and time of exposure have been therefore found to be significant factors. These findings could be used to improve new effective method using SMF in conjunction with the common therapeutic approaches.     

Induction of (2′–5′) oligoadenylate synthetase activity during granulocyte and monocyte differentiation

Summary Variations in the (2′–5′) oligoadenylate synthetase (2–5 A synthetase) level were examined prior to and during the differentiation in culture of the human monocyte cell line U937 and the promyelocytic cell line HL60 in an attempt to reveal whether the enzyme is actively involved in hematopoietic cell maturation. The basal level of this enzyme was much higher in U937 than in HL60 cells. The activity of 2–5 A synthetase was enhanced in both cell lines in response to α, β interferons. During cell differentiation, ten markers were measured. The level of the enzyme rose during the process of cellular maturation in both cell lines. The 2–5 A synthetase activity observed in differentiated HL60 and U937 cells was comparable to that observed in mature normal granulocytes and monocytes respectively. Induction of U937 differentiation by chemicals was associated with detectable production of IFN. The increase in enzyme activity observed was mostly dependent on endogenous production of interferon, since it was inhibited by interferon antibodies. Kinetic studies showed that in U937 cells 2–5 A synthetase was expressed prior to several of the differentiation markers. The rise in the enzyme's level observed during the differentiation of HL60 cells was independent of endogenous production of interferon, since it was not inhibited by the addition of anti-interferon antibodies. These results suggest that different biochemical and molecular mechanisms are responsible for the induction of 2–5 A synthetase observed during the differentiation of hematopoietic cells. In any case, 2–5 A synthetase can be considered as a biochemical marker of cell status and differentiation in hematopoietic cells.     

Antisense CD11b integrin inhibits the development of a differentiated monocyte/macrophage phenotype in human leukemia cells

Macrophage-like development of myeloid leukemia cells which can be induced by agents such as phorbol esters (TPA) is accompanied by integrin expression and cell adhesion. Thus, in differentiating myeloid leukemia cells CD11b is predominantly expressed which can associate with CD18 to form the functional heterodimeric integrin Mac-1. To elucidate the role of cell adhesion during macrophage-like differentiation, we transfected human U937 myeloid leukemia cells with a vector containing the CD11b gene in antisense orientation. Expression of the CD11b antisense gene in stably transfected U937 cells (as-CD11b cells) resulted in an attenuated response to TPA. As-CD11b cells demonstrated poor adhesion to solid substrate upon TPA treatment in contrast to U937 control cells. Constitutive expression of c-myc in as-CD11b transfectants was higher than in control cells and failed to be repressed by TPA treatment. Moreover, unlike control cells, antisense transfectants failed to induce expression of early response genes such as c-jun and the redox factor ref-1 upon TPA stimulation. Consequently, the induction of monocytic differentiation markers such as the activity of alpha-naphthyl acetate esterase, the capacity to reduce nitroblue tetrazolium and the expression of the vimentin gene was much lower in antisense transfectants than in control U937 cells. According to the failure to undergo a monocytic differentiation program, TPA treatment of as-CD11b cells resulted in a progressively increasing amount of apoptotic cells whereas the differentiated population of U937 control cells remained alive. Taken together, these data suggest that the integrin-mediated (particularly CD11b-mediated) adhesion of myeloid leukemia cells in the course of induced monocytic differentiation is crucial for cell attachment, development of a monocytic phenotype and subsequent survival.     

Differentiation of human U937 promonocytic cells is impaired by moderate copper deficiency

Copper (Cu) deficiency suppresses macrophage activities in animals and humans. Our previous studies indicated that the induction of Cu deficiency in differentiated U937 monocytic cells impairs respiratory burst and bactericidal activities and lipopolysaccharide-mediated secretion of inflammatory mediators. The current investigation examined the roles of Cu in the monocytic differentiation process. Human U937 promonocytic cells were exposed to a high affinity Cu chelator (5 microM 2,3,2-tetraamine [tet]) for 24 hr before inducing differentiation by treatment with 1,25-dihydroxyvitamin D3 plus interferon-gamma (DI). This procedure decreased cell Cu by 55% without compromising cellular Zn, Fe, or general metabolic activities. Lower Cu status significantly attenuated the expression of maturation markers Mac-1 (CD11b), ICAM-1 (CD54), and LPS-R (CD14). This change was associated with a marked suppression in respiratory burst activity and killing of Salmonella. To examine if the adverse effect of inadequate Cu on the DI-induced differentiation represented a more general defect, U937 cells were treated with phorbol 12-myristate 13-acetate (PMA). Lower Cu status also suppressed PMA-mediated differentiation of U937 cells. Supplemental Cu, but not Zn or Fe, blocked the tet-induced declines in cell Cu, expression of maturation markers, and respiratory burst and bactericidal activities. These results demonstrate that Cu is essential for the monocytic differentiation process that contributes to the competency of the host's defense system.